Cloning of cytochrome p450 genes from nicotiana

ABSTRACT

The present invention relates to p450 enzymes and nucleic acid sequences encoding p450 enzymes in Nicotiana, and methods of using those enzymes and nucleic acid sequences to alter plant phenotypes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims the benefit of priorityunder 35 U.S.C. §120 to U.S. application Ser. No. 10/934,944 having afiling date of Sep. 3, 2004, which claims the benefit of priority under35 U.S.C. §119(e) of U.S. Application No. 60/503,989, filed Sep. 18,2003, U.S. Provisional Application No. 60/485,368, filed Jul. 8, 2003,and U.S. Provisional Application No. 60/418,933, filed Oct. 16, 2002.This application is also a continuation-in-part of U.S. application Ser.No. 10/686,947, filed Oct. 16, 2003, now abandoned, which is acontinuation-in-part of U.S. application Ser. No. 10/387,346, filed Mar.12, 2003, now abandoned, which is a continuation-in-part of U.S.application Ser. No. 10/340,861, filed Jan. 10, 2003, now abandoned,which is a continuation-in-part of U.S. application Ser. No. 10/293,252,filed Nov. 13, 2002, now abandoned, which claims the benefit of U.S.Provisional Application No. 60/363,684, filed Mar. 12, 2002, U.S.Provisional Application No. 60/347,444, filed Jan. 11, 2002, and U.S.Provisional Application No. 60/337,684, filed on Nov. 13, 2001.

The present invention relates to nucleic acid sequences encodingcytochrome p450 enzymes (hereinafter referred to as p450 and p450enzymes) in Nicotiana plants and methods for using those nucleic acidsequences to alter plant phenotypes.

BACKGROUND

Cytochrome p450s catalyze enzymatic reactions for a diverse range ofchemically dissimilar substrates that include the oxidative,peroxidative and reductive metabolism of endogenous and xenobioticsubstrates. In plants, p450s participate in biochemical pathways thatinclude the synthesis of plant products such as phenylpropanoids,alkaloids, terpenoids, lipids, cyanogenic glycosides, and glucosinolates(Chappel, Annu Rev. Plant Physiol. Plant Mol. Biol. 198, 49:311-343).Cytochrome p450s, also known as p450 heme-thiolate proteins, usually actas terminal oxidases in multi-component electron transfer chains, calledp450-containing monooxygenase systems. Specific reactions catalyzedinclude demethylation, hydroxylation, epoxidation, N-oxidation,sulfooxidation, N-, S-, and O-dealkylations, desulfation, deamination,and reduction of azo, nitro, and N-oxide groups.

The diverse role of Nicotiana plant p450 enzymes has been implicated ineffecting a variety of plant metabolites such as phenylpropanoids,alkaloids, terpenoids, lipids, cyanogenic glycosides, glucosinolates anda host of other chemical entities. During recent years, it is becomingapparent that some p450 enzymes can impact the composition of plantmetabolites in plants. For example, it has been long desired to improvethe flavor and aroma of certain plants by altering its profile ofselected fatty acids through breeding; however very little is knownabout mechanisms involved in controlling the levels of these leafconstituents. The down regulation of p450 enzymes associated with themodification of fatty acids may facilitate accumulation of desired fattyacids that provide more preferred leaf phenotypic qualities. Thefunction of p450 enzymes and their broadening roles in plantconstituents is still being discovered. For instance, a special class ofp450 enzymes was found to catalyze the breakdown of fatty acid intovolatile C6- and C9-aldehydes and -alcohols that are major contributorsof “fresh green” odor of fruits and vegetables. The level of other noveltargeted p450s may be altered to enhance the qualities of leafconstituents by modifying lipid composition and related break downmetabolites in Nicotiana leaf. Several of these constituents in leaf areaffected by senescence that stimulates the maturation of leaf qualityproperties. Still other reports have shown that p450s enzymes are play afunctional role in altering fatty acids that are involved inplant-pathogen interactions and disease resistance.

In other instances, p450 enzymes have been suggested to be involved inalkaloid biosynthesis. Nornicotine is a minor alkaloid found inNicotiana tabaceum. It has been postulated that it is produced by thep450 mediated demethylation of nicotine followed by acylation andnitrosation at the N position thereby producing a series ofN-acylnonicotines and N-nitrosonornicotines. N-demethylation, catalyzedby a putative p450 demethylase, is thought to be a primary source ofnornicotine biosyntheses in Nicotiana. While the enzyme is believed tobe microsomal, thus far a nicotine demethylase enzyme has not beensuccessfully purified, nor have the genes involved been isolated.

Furthermore, it is hypothesized but not proven that the activity of p450enzymes is genetically controlled and also strongly influenced byenvironment factors. For example, the demethylation of nicotine inNicotiana is thought to increase substantially when the plants reach amature stage. Furthermore, it is hypothesized yet not proven that thedemethylase gene contains a transposable element that can inhibittranslation of RNA when present.

The large multiplicity of p450 enzyme forms, their differing structureand function have made their research on Nicotiana p450 enzymes verydifficult before the enclosed invention. In addition, cloning of p450enzymes has been hampered at least in part because thesemembrane-localized proteins are typically present in low abundance andoften unstable to purification. Hence, a need exists for theidentification of p450 enzymes in plants and the nucleic acid sequencesassociated with those p450 enzymes. In particular, only a few cytochromep450 proteins have been reported in Nicotiana. The inventions describedherein entail the discovery of a substantial number of cytochrome p450fragments that correspond to several groups of p450 species based ontheir sequence identity.

SUMMARY

The present invention is directed to plant p450 enzymes. The presentinvention is further directed to plant p450 enzymes from Nicotiana. Thepresent invention is also directed to p450 enzymes in plants whoseexpression is induced by ethylene and/or plant senescence. The presentinvention is yet further directed to nucleic acid sequences in plantshaving enzymatic activities, for example, being categorized asoxygenase, demethylase and the like, or other and the use of thosesequences to reduce or silence the expression or over-expression ofthese enzymes. The invention also relates to p450 enzymes found inplants containing higher nornicotine levels than plants exhibiting lowernornicotine levels.

In one aspect, the invention is directed to nucleic acid sequences asset forth in SEQ. ID. Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183,185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211,213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239,241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267,269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295 and297.

In a second related aspect, those fragments containing greater than 75%identity in nucleic acid sequence were placed into groups dependent upontheir identity in a region corresponding to the first nucleic acidfollowing the cytochrome p450 motif GXRXCX(G/A) (SEQ. ID NO.: 366) tothe stop codon. The representative nucleic acid groups and respectivespecies are shown in Table I.

In a third aspect, the invention is directed to amino acid sequences asset forth in SEQ. ID. Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60,62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184,186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212,214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240,242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268,270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296 and298.

In a fourth related aspect, those fragments containing greater than 71%identity in amino acid sequence were placed into groups dependent upontheir identity to each other in a region corresponding to the firstamino acid following the cytochrome p450 motif GXRXCX(G/A) (SEQ. ID No.:366) to the stop codon. The representative amino acid groups andrespective species are shown in Table II.

In a fifth aspect, the invention is directed to amino acid sequences offull length genes as set forth in SEQ. ID. Nos. 150, 152, 154, 156, 158,160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186,188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214,216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242,244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270,272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296 and 298.

In a sixth related aspect, those full length genes containing 85% orgreater identity in amino acid sequence were placed into groupsdependent upon the identity to each other. The representative amino acidgroups and respective species are shown in Table III.

In a seventh aspect, the invention is directed to amino acid sequencesof the fragments set forth in SEQ. ID. Nos. 299-357.

In the eighth related aspect, those fragments containing 90% or greateridentity in amino acid sequence were placed into groups dependent upontheir identity to each other in a region corresponding to the firstcytochrome p450 domain, UXXRXXZ (SEQ. ID No.: 367), to the thirdcytochrome domain, GXRXO (SEQ. ID No.: 368), where U is E or K, X is anyamino acid and Z is R, T, S or M. The representative amino acid groupsrespective species shown in Table IV.

In a ninth related aspect, the reduction or elimination orover-expression of p450 enzymes in Nicotiana plants may be accomplishedtransiently using RNA viral systems.

Resulting transformed or infected plants are assessed for phenotypicchanges including, but not limited to, analysis of endogenous p450 RNAtranscripts, p450 expressed peptides, and concentrations of plantmetabolites using techniques commonly available to one having ordinaryskill in the art.

In a tenth important aspect, the present invention is also directed togeneration of trangenic Nicotiana lines that have altered p450 enzymeactivity levels. In accordance with the invention, these transgeniclines include nucleic acid sequences that are effective for reducing orsilencing or increasing the expression of certain enzyme thus resultingin phenotypic effects within Nicotiana. Such nucleic acid sequencesinclude SEQ. ID. Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 143, 145, 147, 149, 151, 153, 155, 157, 159,161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187,189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215,217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243,245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271,273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295 and 297.

In a very important eleventh aspect of the invention, plant cultivarsincluding nucleic acids of the present invention in a down regulationcapacity using either full length genes or fragments thereof or in anover-expression capacity using full length genes will have alteredmetabolite profiles relative to control plants.

In a twelfth aspect of the invention, plant cultivars including nucleicacid of the present invention using either full length genes orfragments thereof in modifying the biosynthesis or breakdown ofmetabolites derived from the plant or external to the plants, will haveuse in tolerating certain exogenous chemicals or plant pests. Suchnucleic acid sequences include SEQ ID. Nos. 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87,89, 91, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177,179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205,207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233,235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261,263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289,291, 293, 295 and 297.

In a thirteenth aspect, the present invention is directed to thescreening of plants, more preferably Nicotiana, that contain genes thathave substantial nucleic acid identity to the taught nucleic acidsequence. The use of the invention would be advantageous to identify andselect plants that contain a nucleic acid sequence with exact orsubstantial identity where such plants are part of a breeding programfor traditional or transgenic varieties, a mutagenesis program, ornaturally occurring diverse plant populations. The screening of plantsfor substantial nucleic acid identity may be accomplished by evaluatingplant nucleic acid materials using a nucleic acid probe in conjunctionwith nucleic acid detection protocols including, but not limited to,nucleic acid hybridization and PCR analysis. The nucleic acid probe mayconsist of the taught nucleic acid sequence or fragment thereofcorresponding to SEQ ID 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185,187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213,215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241,243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269,271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295 and 297.

In a fourteenth aspect, the present invention is directed to theidentification of plant genes, more preferably Nicotiana, that sharesubstantial amino acid identity corresponding to the taught nucleic acidsequence. The identification of plant genes including both cDNA andgenomic clones, those cDNAs and genomic clones, more preferably fromNicotiana may be accomplished by screening plant cDNA libraries using anucleic acid probe in conjunction with nucleic acid detection protocolsincluding, but not limited to, nucleic acid hybridization and PCRanalysis. The nucleic acid probe may be comprised of nucleic acidsequence or fragment thereof corresponding to SEQ ID 1, 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 89, 91, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 143, 145 and 147.

In an alternative fifteenth aspect, cDNA expression libraries thatexpress peptides may be screened using antibodies directed to part orall of the taught amino acid sequence. Such amino acid sequences includeSEQ ID 2, 4, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 96, 98, 100, 102, 104, 106,108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134,136, 138, 140, 144, 146, 148.

In a sixteenth important aspect, the present invention is also directedto generation of transgenic Nicotiana lines that have over-expression ofp450 enzyme activity levels. In accordance with the invention, thesetransgenic lines include all nucleic acid sequences encoding the aminoacid sequences of full length genes that are effective for increasingthe expression of certain enzyme thus resulting in phenotypic effectswithin Nicotiana. Such amino acid sequences include SEQ. ID. 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180,182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208,210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236,238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264,266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292,294, 296 and 298.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A shows amino acid identity group members.

FIG. 1B shows amino acid identity group members.

FIG. 1C shows amino acid identity group members.

FIG. 2A shows a comparison of Sequence Groups.

FIG. 2B shows a comparison of Sequence Groups.

FIG. 2C shows a comparison of Sequence Groups.

FIG. 2D shows a comparison of Sequence Groups.

FIG. 2E shows a comparison of Sequence Groups.

FIG. 2F shows a comparison of Sequence Groups.

FIG. 2G shows a comparison of Sequence Groups.

FIG. 2H shows a comparison of Sequence Groups.

FIG. 2I shows a comparison of Sequence Groups.

FIG. 2J shows a comparison of Sequence Groups.

FIG. 3A illustrates alignment of full length clones.

FIG. 3B shows a comparison of Sequence Groups.

FIG. 3C shows a comparison of Sequence Groups.

FIG. 3D shows a comparison of Sequence Groups.

FIG. 3E shows a comparison of Sequence Groups.

FIG. 4 shows a procedure used for cloning of cytochrome p450 cDNAfragments by PCR.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton et al. (1994)Dictionary of Microbiology and Molecular Biology, second edition, JohnWiley and Sons (New York) provides one of skill with a generaldictionary of many of the terms used in this invention. All patents andpublications referred to herein are incorporated by reference herein.For purposes of the present invention, the following terms are definedbelow.

“Enzymatic activity” is meant to include demethylation, hydroxylation,epoxidation, N-oxidation, sulfooxidation, N-, S-, and O-dealkylations,desulfation, deamination, and reduction of azo, nitro, and N-oxidegroups. The term “nucleic acid” refers to a deoxyribonucleotide orribonucleotide polymer in either single- or double-stranded form, orsense or anti-sense, and unless otherwise limited, encompasses knownanalogues of natural nucleotides that hybridize to nucleic acids in amanner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence includes the complementarysequence thereof.

The terms “operably linked”, “in operable combination”, and “in operableorder” refer to functional linkage between a nucleic acid expressioncontrol sequence (such as a promoter, signal sequence, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence affects transcription and/ortranslation of the nucleic acid corresponding to the second sequence.

The term “recombinant” when used with reference to a cell indicates thatthe cell replicates a heterologous nucleic acid, expresses said nucleicacid or expresses a peptide, heterologous peptide, or protein encoded bya heterologous nucleic acid. Recombinant cells can express genes or genefragments in either the sense or antisense form that are not foundwithin the native (non-recombinant) form of the cell. Recombinant cellscan also express genes that are found in the native form of the cell,but wherein the genes are modified and re-introduced into the cell byartificial means.

A “structural gene” is that portion of a gene comprising a DNA segmentencoding a protein, polypeptide or a portion thereof, and excluding the5′ sequence which drives the initiation of transcription. The structuralgene may alternatively encode a nontranslatable product. The structuralgene may be one which is normally found in the cell or one which is notnormally found in the cell or cellular location wherein it isintroduced, in which case it is termed a “heterologous gene”. Aheterologous gene may be derived in whole or in part from any sourceknown to the art, including a bacterial genome or episome, eukaryotic,nuclear or plasmid DNA, cDNA, viral DNA or chemically synthesized DNA. Astructural gene may contain one or more modifications that could effectbiological activity or its characteristics, the biological activity orthe chemical structure of the expression product, the rate of expressionor the manner of expression control. Such modifications include, but arenot limited to, mutations, insertions, deletions and substitutions ofone or more nucleotides. The structural gene may constitute anuninterrupted coding sequence or it may include one or more introns,bounded by the appropriate splice junctions. The structural gene may betranslatable or non-translatable, including in an anti-senseorientation. The structural gene may be a composite of segments derivedfrom a plurality of sources and from a plurality of gene sequences(naturally occurring or synthetic, where synthetic refers to DNA that ischemically synthesized).

“Derived from” is used to mean taken, obtained, received, traced,replicated or descended from a source (chemical and/or biological). Aderivative may be produced by chemical or biological manipulation(including, but not limited to, substitution, addition, insertion,deletion, extraction, isolation, mutation and replication) of theoriginal source.

“Chemically synthesized”, as related to a sequence of DNA, means thatportions of the component nucleotides were assembled in vitro. Manualchemical synthesis of DNA may be accomplished using well establishedprocedures (Caruthers, Methodology of DNA and RNA Sequencing, (1983),Weissman (ed.), Praeger Publishers, New York, Chapter 1); automatedchemical synthesis can be performed using one of a number ofcommercially available machines.

Optimal alignment of sequences for comparison may be conducted by thelocal homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman and Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearsonand Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al.,1990) is available from several sources, including the National Centerfor Biological Information (NCBI, Bethesda, Md.) and on the Internet,for use in connection with the sequence analysis programs blastp,blastn, blastx, tblastn and tblastx. It can be accessed atncbi.nlm.nih.gov/BLAST/. A description of how to determine sequenceidentity using this program is available atncbi.nlm.nih.gov/BLAST/blast_help.html.

The terms “substantial amino acid identity” or “substantial amino acidsequence identity” as applied to amino acid sequences and as used hereindenote a characteristic of a polypeptide, wherein the peptide comprisesa sequence that has at least 70 percent sequence identity, preferably 80percent amino acid sequence identity, more preferably 90 percent aminoacid sequence identity, and most preferably at least 99 to 100 percentsequence identity as compared to a reference group over regioncorresponding to the first amino acid following the cytochrome p450motif GXRXCX(G/A) (SEQ. ID No.: 366) to the stop codon of the translatedpeptide.

The terms “substantial nucleic acid identity” or “substantial nucleicacid sequence identity” as applied to nucleic acid sequences and as usedherein denote a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 75 percentsequence identity, preferably 81 percent sequence identity, morepreferably at least 91 percent sequence identity, and most preferably atleast 99 to 100 percent sequence identity as compared to a referencegroup over region corresponding to the first nucleic acid following thecytochrome p450 motif GXRXCX (G/A) (SEQ. ID No.: 366) to the stop codonof the translated peptide.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Generally, stringent conditions are selected tobe about 5° C. to about 20° C., usually about 10° C. to about 15° C.,lower than the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. The Tm is the temperature (under definedionic strength and pH) at which 50% of the target sequence hybridizes toa matched probe. Typically, stringent conditions will be those in whichthe salt concentration is about 0.02 molar at pH 7 and the temperatureis at least about 60° C. For instance in a standard Southernhybridization procedure, stringent conditions will include an initialwash in 6×SSC at 42° C. followed by one or more additional washes in0.2×SSC at a temperature of at least about 55° C., typically about 60°C. and often about 65° C.

Nucleotide sequences are also substantially identical for purposes ofthis invention when the polypeptides and/or proteins which they encodeare substantially identical. Thus, where one nucleic acid sequenceencodes essentially the same polypeptide as a second nucleic acidsequence, the two nucleic acid sequences are substantially identical,even if they would not hybridize under stringent conditions due todegeneracy permitted by the genetic code (see, Darnell et al. (1990)Molecular Cell Biology, Second Edition Scientific American Books W. H.Freeman and Company New York for an explanation of codon degeneracy andthe genetic code). Protein purity or homogeneity can be indicated by anumber of means well known in the art, such as polyacrylamide gelelectrophoresis of a protein sample, followed by visualization uponstaining. For certain purposes high resolution may be needed and HPLC ora similar means for purification may be utilized.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) into a cell. A vector may act toreplicate DNA and may reproduce independently in a host cell. The term“vehicle” is sometimes used interchangeably with “vector.” The term“expression vector” as used herein refers to a recombinant DNA moleculecontaining a desired coding sequence and appropriate nucleic acidsequences necessary for the expression of the operably linked codingsequence in a particular host organism. Nucleic acid sequences necessaryfor expression in prokaryotes usually include a promoter, an operator(optional), and a ribosome binding site, often along with othersequences. Eucaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

For the purpose of regenerating complete genetically engineered plantswith roots, a nucleic acid may be inserted into plant cells, forexample, by any technique such as in vivo inoculation or by any of theknown in vitro tissue culture techniques to produce transformed plantcells that can be regenerated into complete plants. Thus, for example,the insertion into plant cells may be by in vitro inoculation bypathogenic or non-pathogenic A. tumefaciens. Other such tissue culturetechniques may also be employed.

“Plant tissue” includes differentiated and undifferentiated tissues ofplants, including, but not limited to, roots, shoots, leaves, pollen,seeds, tumor tissue and various forms of cells in culture, such assingle cells, protoplasts, embryos and callus tissue. The plant tissuemay be in planta or in organ, tissue or cell culture.

“Plant cell” as used herein includes plant cells in planta and plantcells and protoplasts in culture.

“cDNA” or “complementary DNA” generally refers to a single stranded DNAmolecule with a nucleotide sequence that is complementary to an RNAmolecule. cDNA is formed by the action of the enzyme reversetranscriptase on an RNA template.

Strategies for Obtaining Nucleic Acid Sequences

In accordance with the present invention, RNA was extracted fromNicotiana tissue of converter and non

converter Nicotiana lines. The extracted RNA was then used to createcDNA. Nucleic acid sequences of the present invention were thengenerated using two strategies.

In the first strategy, the poly A enriched RNA was extracted from planttissue and cDNA was made by reverse transcription PCR. The single strandcDNA was then used to create p450 specific PCR populations usingdegenerate primers plus a oligo d(T) reverse primer. The primer designwas based on the highly conserved motifs of p450. Sequence fragmentsfrom plasmids containing appropriate size inserts were further analyzed.These size inserts typically ranged from about 300 to about 800nucleotides depending on which primers were used.

In a second strategy, a cDNA library was initially constructed. The cDNAin the plasmids was used to create p450 specific PCR populations usingdegenerate primers plus T7 primer on plasmid as reverse primer. As inthe first strategy, sequence fragments from plasmids containingappropriate size inserts were further analyzed.

Nicotiana plant lines known to produce high levels of nornicotine(converter) and plant lines having undetectable levels of nornicotinemay be used as starting materials.

Leaves can then be removed from plants and treated with ethylene toactivate p450 enzymatic activities defined herein. Total RNA isextracted using techniques known in the art. cDNA fragments can then begenerated using PCR (RT-PCR) with the oligo d(T) primer as described inFIG. 4. The cDNA library can then be constructed more fully described inexamples herein.

The conserved region of p450 type enzymes can be used as a template fordegenerate primers (FIG. 4). Using degenerate primers, p450 specificbands can be amplified by PCR. Bands indicative for p450 like enzymescan be identified by DNA sequencing. PCR fragments can be characterizedusing BLAST search, alignment or other tools to identify appropriatecandidates.

Sequence information from identified fragments can be used to developPCR primers. These primers in combination of plasmid primers in cDNAlibrary were used to clone full length p450 genes. Large-scale Southernreverse analysis was conducted to examine the differential expressionfor all fragment clones obtained and in some cases full length clones.In this aspect of the invention, these large-scale reverse Southernassays can be conducted using labeled total cDNA's from differenttissues as a probe to hybridize with cloned DNA fragments in order toscreen all cloned inserts.

Nonradioactive and radioactive (P32) Northern blotting assays were alsoused to characterize clones p450 fragments and full length clones.

Peptide specific antibodies were made against several full-length clonesby deriving their amino acid sequence and selecting peptide regions thatwere antigenic and unique relative to other clones. Rabbit antibodieswere made to synthetic peptides conjugated to a carrier protein. Westernblotting analyses or other immunological methods were performed on planttissue using these antibodies.

Nucleic acid sequences identified as described above can be examined byusing virus induced gene silencing technology (VIGS, Baulcombe, CurrentOpinions in Plant Biology, 1999, 2:109-113).

Peptide specific antibodies were made for several full-length clones byderiving their amino acid sequence and selecting peptide regions thatwere potentially antigenic and were unique relative to other clones.Rabbit antibodies were made to synthetic peptides conjugated to acarrier protein. Western blotting analyses were performed using theseantibodies.

In another aspect of the invention, interfering RNA technology (RNAi) isused to further characterize cytochrome p450 enzymatic activities inNicotiana plants of the present invention. The following referenceswhich describe this technology are incorporated by reference herein,Smith et al., Nature, 2000, 407:319-320; Fire et al., Nature, 1998,391:306-311; Waterhouse et al., PNAS, 1998, 95:13959-13964; Stalberg etal., Plant Molecular Biology, 1993, 23:671-683; Baulcombe, CurrentOpinions in Plant Biology, 1999, 2:109-113; and Brigneti et al., EMBOJournal, 1998, 17(22):6739-6746. Plants may be transformed using RNAitechniques, antisense techniques, or a variety of other methodsdescribed.

Several techniques exist for introducing foreign genetic material intoplant cells, and for obtaining plants that stably maintain and expressthe introduced gene. Such techniques include acceleration of geneticmaterial coated onto microparticles directly into cells (U.S. Pat. Nos.4,945,050 to Cornell and 5,141,131 to DowElanco). Plants may betransformed using Agrobacterium technology, see U.S. Pat. No. 5,177,010to University of Toledo, 5,104,310 to Texas A&M, European PatentApplication 013162481, European Patent Applications 120516, 15941881,European Patent Applications 120516, 159418B1 and 176,112 toSchilperoot, U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763 and4,940,838 and 4,693,976 to Schilperoot, European Patent Applications116718, 290799, 320500 all to MaxPlanck, European Patent Applications604662 and 627752 to Japan Nicotiana, European Patent Applications0267159, and 0292435 and U.S. Pat. No. 5,231,019 all to Ciba Geigy, U.S.Pat. Nos. 5,463,174 and 4,762,785 both to Calgene, and U.S. Pat. Nos.5,004,863 and 5,159,135 both to Agracetus. Other transformationtechnology includes whiskers technology, see U.S. Pat. Nos. 5,302,523and 5,464,765 both to Zeneca. Electroporation technology has also beenused to transform plants, see WO 87/06614 to Boyce Thompson Institute,5,472,869 and 5,384,253 both to Dekalb, WO9209696 and WO9321335 both toPGS. All of these transformation patents and publications areincorporated by reference. In addition to numerous technologies fortransforming plants, the type of tissue which is contacted with theforeign genes may vary as well. Such tissue would include but would notbe limited to embryogenic tissue, callus tissue type I and II,hypocotyl, meristem, and the like. Almost all plant tissues may betransformed during dedifferentiation using appropriate techniques withinthe skill of an artisan.

Foreign genetic material introduced into a plant may include aselectable marker. The preference for a particular marker is at thediscretion of the artisan, but any of the following selectable markersmay be used along with any other gene not listed herein which couldfunction as a selectable marker. Such selectable markers include but arenot limited to aminoglycoside phosphotransferase gene of transposon Tn5(Aph II) which encodes resistance to the antibiotics kanamycin, neomycinand G418, as well as those genes which code for resistance or toleranceto glyphosate; hygromycin; methotrexate; phosphinothricin (bar);imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such aschlorosulfuron; bromoxynil, dalapon and the like.

In addition to a selectable marker, it may be desirous to use a reportergene. In some instances a reporter gene may be used without a selectablemarker. Reporter genes are genes which are typically not present orexpressed in the recipient organism or tissue. The reporter genetypically encodes for a protein which provide for some phenotypic changeor enzymatic property. Examples of such genes are provided in K. Weisinget al. Ann. Rev. Genetics, 22, 421 (1988), which is incorporated hereinby reference. Preferred reporter genes include without limitationglucuronidase (GUS) gene and GFP genes.

Once introduced into the plant tissue, the expression of the structuralgene may be assayed by any means known to the art, and expression may bemeasured as mRNA transcribed, protein synthesized, or the amount of genesilencing that occurs (see U.S. Pat. No. 5,583,021 which is herebyincorporated by reference). Techniques are known for the in vitroculture of plant tissue, and in a number of cases, for regeneration intowhole plants (EP Appln No. 88810309.0). Procedures for transferring theintroduced expression complex to commercially useful cultivars are knownto those skilled in the art.

Once plant cells expressing the desired level of p450 enzyme areobtained, plant tissues and whole plants can be regenerated therefromusing methods and techniques well-known in the art. The regeneratedplants are then reproduced by conventional means and the introducedgenes can be transferred to other strains and cultivars by conventionalplant breeding techniques.

The following examples illustrate methods for carrying out the inventionand should be understood to be illustrative of, but not limiting upon,the scope of the invention which is defined in the appended claims.

EXAMPLES Example I Development of Plant Tissue and Ethylene TreatmentPlant Growth

Plants were seeded in pots and grown in a greenhouse for 4 weeks. The 4week old seedlings were transplanted into individual pots and grown inthe greenhouse for 2 months. The plants were watered 2 times a day withwater containing 150 ppm NPK fertilizer during growth. The expandedgreen leaves were detached from plants to do the ethylene treatmentdescribed below.

Cell Line 78379

Tobacco line 78379, which is a burley tobacco line released by theUniversity of Kentucky was used as a source of plant material. Onehundred plants were cultured as standard in the art of growing tobaccoand transplanted and tagged with a distinctive number (1-100).Fertilization and field management were conducted as recommended.

Three quarters of the 100 plants converted between 20 and 100% of thenicotine to nornicotine. One quarter of the 100 plants converted lessthan 5% of the nicotine to nornicotine. Plant number 87 had the leastconversion (2%) while plant number 21 had 100% conversion. Plantsconverting less than 3% were classified as non-converters.Self-pollinated seed of plant number 87 and plant number 21, as well ascrossed (21×87 and 87×21) seeds were made to study genetic andphenotypic differences. Plants from selfed 21 were converters, and 99%of selfs from 87 were non-converters. The other 1% of the plants from 87showed low conversion (5-15%) Plants from reciprocal crosses were allconverters.

Cell Line 4407

Nicotiana line 4407, which is a burley line was used as a source ofplant material. Uniform and representative plants (100) were selectedand tagged. Of the 100 plants 97 were non-converters and three wereconverters. Plant number 56 had the least amount of conversion (1.2%)and plant number 58 had the highest level of conversion (96%).Self-pollenated seeds and crossed seeds were made with these two plants.

Plants from selfed-58 segregated with 3:1 converter to non-converterratio. Plants 58-33 and 58-25, were identified as homozygous converterand nonconverter plant lines, respectively. The stable conversion of58-33 was confirmed by analysis of its progenies of next generation.

Cell Line PBLBO1

PBLBO1 is a burley line developed by ProfiGen, Inc. and was used as asource of plant material. The converter plant was selected fromfoundation seeds of PBLB01.

Ethylene Treatment Procedures

Green leaves were detached from 2-3 month greenhouse grown plants andsprayed with 0.3% ethylene solution (Prep brand Ethephon(Rhone-Poulenc)). Each sprayed leaf was hung in a curing rack equippedwith humidifier and covered with plastic. During the treatment, thesample leaves were periodically sprayed with the ethylene solution.Approximately 24-48 hour post ethylene treatment, leaves were collectedfor RNA extraction. Another sub-sample was taken for metabolicconstituent analysis to determine the concentration of leaf metabolitesand more specific constituents of interest such as a variety ofalkaloids.

As an example, alkaloids analysis could be performed as follows. Samples(0.1 g) were shaken at 150 rpm with 0.5 ml 2N NaOH, and a 5 mlextraction solution which contained quinoline as an internal standardand methyl t-butyl ether. Samples were analyzed on a HP 6890 GC equippedwith a FID detector. A temperature of 250° C. was used for the detectorand injector. An HP column (30 m-0.32 nm-l·m) consisting of fused silicacrosslinked with 5% phenol and 95% methyl silicon was used at atemperature gradient of 110-185° C. at 10° C. per minute. The column wasoperated at 100° C. with a flow rate of 1.7 cm³ min⁻¹ with a split ratioof 40:1 with a 2.1 injection volume using helium as the carrier gas.

Example 2 RNA Isolation

For RNA extractions, middle leaves from 2 month old greenhouse grownplants were treated with ethylene as described. The 0 and 24-48 hourssamples were used for RNA extraction. In some cases, leaf samples underthe senescence process were taken from the plants 10 days postflower-head removal. These samples were also used for extraction. TotalRNA was isolated using Rneasy Plant Mini Kit® (Qiagen, Inc., Valencia,Calif.) following manufacturer's protocol.

The tissue sample was ground under liquid nitrogen to a fine powderusing a DEPC treated mortar and pestle. Approximately 100 milligrams ofground tissue were transferred to a sterile 1.5 ml eppendorf tube. Thissample tube was placed in liquid nitrogen until all samples werecollected. Then, 450μ-l of Buffer RLT as provided in the kit (with theaddition of Mercaptoethanol) was added to each individual tube. Thesample was vortexed vigorously and incubated at 56° C. for 3 minutes.The lysate was then, applied to the QIAshredder™ spin column sitting ina 2-ml collection tube, and centrifuged for 2 minutes at maximum speed.The flow through was collected and 0.5 volume of ethanol was added tothe cleared lysate. The sample is mixed well and transferred to anRneasy® mini spin column sitting in a 2 ml collection tube. The samplewas centrifuged for 1 minute at 10,000 rpm. Next, 700 μl of buffer RW1was pipetted onto the Rneasy® column and centrifuged for 1 minute at10,000 rpm. Buffer RPE was pipetted onto the Rneasy® column in a newcollection tube and centrifuged for 1 minute at 10,000 rpm. Buffer RPEwas again, added to the Rneasy® spin column and centrifuged for 2minutes at maximum speed to dry the membrane. To eliminate any ethanolcarry over, the membrane was placed in a separate collection tube andcentrifuged for an additional 1 minute at maximum speed. The Rneasy®column was transferred into a new 1.5 ml collection tube, and 40 μl ofRnase-free water was pipetted directly onto the Rneasy® membrane. Thisfinal elute tube was centrifuged for 1 minute at 10,000 rpm. Quality andquantity of total RNA was analyzed by denatured formaldehyde gel andspectrophotometer.

Poly (A) RNA was isolated using Oligotex™ poly A+ RNA purification kit(Qiagen Inc.) following manufacture's protocol. About 200 μg total RNAin 250 μl maximum volume was used. A volume of 250 μl of Buffer OBB and15 μl of Oligotex™ suspension was added to the 250 μl of total RNA. Thecontents were mixed thoroughly by pipetting and incubated for 3 minutesat 70° C. on a heating block. The sample was then, placed at roomtemperature for approximately 20 minutes. The oligotex:mRNA complex waspelleted by centrifugation for 2 minutes at maximum speed. All but 50 μlof the supernatant was removed from the microcentrifuge tube. The samplewas treated further by OBB buffer. The oligotex:mRNA pellet wasresuspended in 400 μl of Buffer OW2 by vortexing. This mix wastransferred onto a. small spin column placed in a new tube andcentrifuged for 1 minute at maximum speed.

The spin column was transferred to a new tube and an additional 400 μlof Buffer OW2 was added to the column. The tube was then centrifuged for1 minute at maximum speed. The spin column was transferred to a final1.5 ml microcentrifuge tube. The sample was eluted with 60 ul of hot(70° C.) Buffer OEB. Poly A product was analyzed by denaturedformaldehyde gels and spectrophotometric analysis.

Example 3 Reverse Transcription-PCR

First strand cDNA was produced using SuperScript reverse transcriptasefollowing manufacturer's protocol (Invitrogen, Carlsbad, Calif.). Thepoly A+ enriched RNA/oligo dT primer mix consisted of less than 5 μg oftotal RNA, 1 μl of 10 mM dNTP mix, 1 μl of Oligo d(T)₁₂₋₁₈ (0.5 μg/μl),and up to 10 μl of DEPC-treated water. Each sample was incubated at 65°C. for 5 minutes, then placed on ice for at least 1 minute. A reactionmixture was prepared by adding each of the following components inorder: 2 μl 10×RT buffer, 4 μA of 25 mM MgCl₂, 2 μl of 0.1 M DTT, and 1μl of RNase OUT Recombinant RNase Inhibitor. An addition of 9 μl ofreaction mixture was pipetted to each RNA/primer mixture and gentlymixed. It was incubated at 42° C. for 2 minutes and 1 μl of Super ScriptII™ RT was added to each tube. The tube was incubated for 50 minutes at42° C. The reaction was terminated at 70° C. for 15 minutes and chilledon ice. The sample was collected by centrifugation and 1 μl of RNase Hwas added to each tube and incubated for 20 minutes at 37° C. The secondPCR was carried out with 200 pmoles of forward primer (degenerateprimers as in SEQ.ID Nos. 358-362) and 100 pmoles reverse primer (mix of18 nt oligo d(T) followed by 1 random base).

Reaction conditions were 94° C. for 2 minutes and then performed 40cycles of PCR at 94° C. for 1 minute, 45° to 60° C. for 2 minutes, 72°C. for 3 minutes with a 72° C. extension for an extra 10 min.

Ten microliters of the amplified sample were analyzed by electrophoresisusing a 1% agarose gel. The correct size fragments were purified fromagarose gel.

Example 4 Generation of PCR Fragment Populations

PCR fragments from Example 3 were ligated into a pGEM-T® Easy Vector(Promega, Madison, Wis.) following manufacturer's instructions. Theligated product was transformed into JM109 competent cells and plated onLB media plates for blue/white selection. Colonies were selected andgrown in a 96 well plate with 1.2 ml of LB media overnight at 37° C.Frozen stock was generated for all selected colonies. Plasmid DNA fromplates were purified using Beckman's Biomeck 2000 miniprep robotics withWizard SV Miniprep® kit (Promega). Plasmid DNA was eluted with 100μl-water and stored in a 96 well plate. Plasmids were digested by EcoR1and were analyzed using 1% agarose gel to confirm the DNA quantity andsize of inserts. The plasmids containing a 400-600 bp insert weresequenced using an CEQ 2000 sequencer (Beckman, Fullerton, Calif.). Thesequences were aligned with GenBank database by BLAST search. The p450related fragments were identified and further analyzed. Alternatively,p450 fragments were isolated from substraction libraries. Thesefragments were also analyzed as described above.

Example 5 Construction of cDNA Library

A cDNA library was constructed by preparing total RNA from ethylenetreated leaves as follows. First, total RNA was extracted from ethylenetreated leaves of tobacco line 58-33 using a modified acid phenol andchloroform extraction protocol. Protocol was modified to use one gram oftissue that was ground and subsequently vortexed in 5 ml of extractionbuffer (100 mM Tris-HCl, pH 8.5; 200 mM NaCl; 10 mM EDTA; 0.5% SDS) towhich 5 ml phenol (pH5.5) and 5 ml chloroform was added. The extractedsample was centrifuged and the supernatant was saved. This extractionstep was repeated 2-3 more times until the supernatant appeared clear.Approximately 5 ml of chloroform was added to remove trace amounts ofphenol. RNA was precipitated from the combined supernatant fractions byadding a 3-fold volume of ETOH and 1/10 volume of 3M NaOAc (pH5.2) andstoring at −20° C. for 1 hour. After transferring to a Corex glasscontainer the RNA fraction was centrifuged at 9,000 RPM for 45 minutesat 4° C. The pellet was washed with 70% ethanol and spun for 5 minutesat 9,000 RPM at 4° C. After drying the pellet, the pelleted RNA wasdissolved in 0.5 ml RNase free water. The pelleted RNA was dissolved in0.5 ml RNase free water. The quality and quantity of total RNA wasanalyzed by denatured formaldehyde gel and spectrophotometer,respectively.

The resultant total RNA was isolated for poly A+ RNA using an Oligo(dT)cellulose protocol (Invitrogen) and Microcentrifuge spin columns(Invitrogen) by the following protocol. Approximately twenty mg of totalRNA was subjected to twice purification to obtain high quality poly A+RNA. Poly A+ RNA product was analyzed by performing denaturedformaldehyde gel and subsequent RT-PCR of known full-length genes toensure high quality of mRNA.

Next, poly A+ RNA was used as template to produce a cDNA libraryemploying cDNA synthesis kit, ZAP-cDNA® synthesis kit, and ZAP-cDNA®Gigapack® III goldcloning kit (Stratagene, La Jolla, Calif.). The methodinvolved following the manufacture's protocol as specified.Approximately 8 μg of poly A+ RNA was used to construct cDNA library.Analysis of the primary library revealed about 2.5×106-1×107 pfu. Aquality background test of the library was completed by complementationassays using IPTG and X-gal, where recombinant plaques was expressed atmore than 100-fold above the background reaction.

A more quantitative analysis of the library by random PCR showed thataverage size of insert cDNA was approximately 1.2 kb. The method used atwo-step PCR method as followed. For the first step, reverse primerswere designed based on the preliminary sequence information obtainedfrom p450 fragments. The designed reverse primers and T3 (forward)primers were used amplify corresponding genes from the cDNA library. PCRreactions were subjected to agarose electrophoresis and thecorresponding bands of high molecular weight were excised, purified,cloned and sequenced. In the second step, new primers designed from5′UTR or the start coding region of p450 as the forward primers togetherwith the reverse primers (designed from 3′UTR of p450) were used in thesubsequent PCR to obtain full-length p450 clones.

The p450 fragments were generated by PCR amplification from theconstructed cDNA library as described in Example 3 with the exception ofthe reverse primer. The T7 primer located on the plasmid downstream ofcDNA inserts (see FIG. 4) was used as a reverse primer. PCR fragmentswere isolated, cloned and sequenced as described in Example 4.

Full-length p450 genes were isolated by PCR method from constructed cDNAlibrary. Gene specific reverse primers (designed from the downstreamsequence of p450 fragments) and a forward primer (T3 on library plasmid)were used to clone the full length genes. PCR fragments were isolated,cloned and sequenced. If necessary, second step PCR was applied. In thesecond step, new forward primers designed from 5′UTR of cloned p450stogether with the reverse primers designed-from 3′UTR of p450 cloneswere used in the subsequent PCR reactions to obtain full-length p450clones. The clones were subsequently sequenced.

Example 6 Characterization of Cloned Fragments—Reverse Southern BlottingAnalysis

Nonradioactive large scale reverse southern blotting assays wereperformed on all p450 clones identified in above examples to detect thedifferential expression. It was observed that the level of expressionamong different p450 clusters was very different. Further real timedetection was conducted on those with high expression.

Nonradioactive Southern blotting procedures were conducted as follows.

1) Total RNA was extracted from ethylene treated and nontreatedconverter (58-33) and-nonconverter (58

25) leaves using the Qiagen Rnaeasy kit as described in Example 2.

2) Probe was produced by biotin-tail labeling a single strand cDNAderived from poly A+ enriched RNA generated in above step. This labeledsingle strand cDNA was generated by RT-PCR of the converter andnonconverter total RNA (Invitrogen) as described in Example 3 with theexception of using biotinalyted oligo dT as a primer (Promega). Thesewere used as a probe to hybridize with cloned DNA.

3) Plasmid DNA was digested with restriction enzyme EcoR1 and run onagarose gels. Gels were simultaneously dried and transferred to twonylon membranes (Biodyne B®). One membrane was hybridized with converterprobe and the other with nonconverter probe. Membranes wereUV-crosslinked (auto crosslink setting, 254 nm, Stratagene,Stratalinker) before hybridization.

Alternatively, the inserts were PCR amplified from each plasmid usingthe sequences located on both arms of p-GEM plasmid, T3 and SP6, asprimers. The PCR products were analyzed by running on a 96 well Ready

to-run agarose gels. The confirmed inserts were dotted on two nylonmembranes. One membrane was hybridized with converter probe and theother with nonconverter probe.

4) The membranes were hybridized and washed following manufacture'sinstruction with the modification of washing stringency (Enzo MaxSence™kit, Enzo Diagnostics, Inc, Farmingdale, N.Y.). The membranes wereprehybridized with hybridization buffer (2×SSC buffered formamide,containing detergent and hybridization enhancers) at 42° C. for 30 minand hybridized with 10 μl denatured probe overnight at 42° C. Themembranes then were washed in 1× hybridization wash buffer 1 time atroom temperature for 10 min and 4 times at 68° C. for 15 min. Themembranes were ready for the detection.

5) The washed membranes were detected by alkaline phosphatase labelingfollowed by NBT/BCIP colometric detection as described in manufacture'sdetection procedure (Enzo Diagnostics, Inc.). The membranes were blockedfor one hour at room temperature with 1× blocking solution, washed 3times with 1× detection reagents for 10 min, washed 2 times with 1×predevelopment reaction buffer for 5 min and then developed the blots indeveloping solution for 30-45 min until the dots appear. All reagentswere provided by manufacture (Enzo Diagnostics, Inc). In Addition, largescale reverse Southern assay was also performed using KPL southernhybridization and detection Kit™ following manfacturer'sinstruction(KPL, Gaithersburg, Md.).

Example 7 Characterization of Clones—Northern Blot Analysis

Alternative to Southern Blot analysis, some membranes were hybridizedand detected as described in the example of Northern blotting assays.Northern Hybridization was used to detect mRNA differentially expressedin Nicotiana as follows.

A random priming method was used to prepare probes from cloned p450(Megaprime™ DNA Labelling Systems, Amersham Biosciences).

The following components were mixed: 25 ng denatured DNA template; 4 ulof each unlabeled dTTP, dGTP and dCTP; 5 ul of reaction buffer;P³²-labelled dATP and 2 ul of Klenow I; and H₂O, to bring the reactionto 50 μl. The mixture was incubated in 37° C. for 1-4 hours, thenstopped with 2 μl of 0.5 M EDTA. The probe was denatured by incubatingat 95° C. for 5 minutes before use.

RNA samples were prepared from ethylene treated and non-treated freshleaves of several pairs of tobacco lines. In some cases poly A+ enrichedRNA was used. Approximately 15 μg total RNA or 1.8 μg mRNA (methods ofRNA and mRNA extraction as described in Example 5) were brought to equalvolume with DEPC H₂O (5-10 μl). The same volume of loading buffer(1×MOPS; 18.5% Formaldehyde; 50% Formamide; 4% Ficol1400;Bromophenolblue) and 0.5 μl EtBr (0.5 μg/μl) were added. The sampleswere subsequently denatured in preparation for separation of the RNA byelectrophoresis.

Samples were subjected to electrophoresis on a formaldehyde gel (1%Agarose, 1×MOPS, 0.6 M Formaldehyde) with 1×MOP buffer (0.4 MMorpholinopropanesulfonic acid; 0.1 M Na-acetate-3×H2O; 10 mM EDTA;adjust to pH 7.2 with NaOH). RNA was transferred to a Hybond-N+ membrane(Nylon, Amersham Pharmacia Biotech) by capillary method in 10×SSC buffer(1.5 M NaCl; 0.15 M Na-citrate) for 24 hours. Membranes with RNA sampleswere UV-crosslinked (auto crosslink setting, 254 nm, Stratagene,Stratalinker) before hybridization.

The membrane was prehybridized for 1-4 hours at 42° C. with 5-10 mlprehybridization buffer (5×SSC; 50% Formamide; 5×Denhardt's-solution; 1%SDS; 100 μg/ml heat-denatured sheared non-homologous DNA). Oldprehybridization buffer was discarded, and new prehybridization bufferand probe were added. The hybridization was carried out over night at42° C. The membrane was washed for 15 minutes with 2×SSC at roomtemperature, followed by a wash with 2×SSC.

A major focus of the invention was the discovery of novel genes that maybe induced as a result of ethylene treatment or play a key role intobacco leaf quality and constituents. As illustrated in the tablebelow, Northern blots and reverse Southern Blot were useful indetermining which genes were induced by ethylene treatment relative tonon-induced plants. Interestingly, not all fragments were affectedsimilarly in the converter and nonconverter. The cytochrome p450fragments of interest were partially sequenced to determine theirstructural relatedness. This information was used to subsequentlyisolate and characterize full length gene clones of interest.

Induced mRNA Expression Ethylene Treatment Fragments Converter D56-AC7(SEQ ID NO: 35) + D56-AG11 (SEQ ID NO: 31) + D56-AC12 (SEQ ID NO: 45) +D70A-AB5 (SEQ ID NO: 95) + D73-AC9 (SEQ ID NO: 43) + D70A-AA12 (SEQ IDNO: 131) + D73A-AG3 (SEQ ID NO: 129) + D34-52 (SEQ ID NO: 61) + D56-AG6(SEQ ID NO: 51) +

Northern analysis was performed using full length clones on tobaccotissue obtained from converter and nonconverter burley lines that wereinduced by ethylene treatment. The purpose was to identify those fulllength clones that showed elevated expression in ethylene inducedconverter lines relative to ethylene induced converter lines relative toethylene induced nonconverter burley lines. By so doing, thefunctionality relationship of full length clones may be determined bycomparing biochemical differences in leaf constituents between converterand nonconverter lines. As shown in table below, six clones showedsignificantly higher expression, as denoted by ++ and +++, in converterethylene treated tissue than that of nonconverter treated tissue,denoted by +. All of these clones showed little or no expression inconverter and nonconverter lines that were not ethylene treated.

Full Length Clones Converter Nonconverter D101-BA2 ++ + D207-AA5 ++ +D208-AC8 +++ + D237-AD1 ++ + D89-AB1 ++ + ID90A-BB3 ++ +

Example 8 Immunodetection of 1D450S Encoded by the Cloned Genes

Peptide regions corresponding to 20-22 amino acids in length from threep450 clones were selected for 1) having lower or no homology to otherclones and 2) having good hydrophilicity and antigenicity. The aminoacid sequences of the peptide regions selected from the respective p450clones are listed below. The synthesized peptides were conjugated withKHL and then injected into rabbits. Antisera were collected 2 and 4weeks after the 4th injection (Alpha Diagnostic Intl. Inc. San Antonio,Tex.).

D234-AD1 DIDGSKSKLVKAHRKIDEILG (SEQ. ID No.: 369) D90a-BB3RDAFREKETFDENDVEELNY (SEQ. ID No.: 370) D89-AB1 FKNNGDEDRHFSQKLGDLADKY(SEQ. ID No.: 371)

Antisera were examined for crossreactivity to target proteins fromtobacco plant tissue by Western Blot analysis. Crude protein extractswere obtained from ethylene treated (0 to 40 hours) middle leaves ofconverter and nonconverter lines. Protein concentrations of the extractswere determined using RC DC Protein Assay Kit (BIO-RAD) following themanufacturer's protocol.

Two micrograms of protein were loaded onto each lane and the proteinsseparated on 10%-20% gradient gels using the Laemmli SDS-PAGE system.The proteins were transferred from gels to PROTRAN® NitrocelluloseTransfer Membranes (Schleicher & Schuell) with the Trans-Blot® Semi-Drycell (BIO-RAD). Target p450 proteins were detected and visualized withthe ECL Advance” Western Blotting Detection Kit (Amersham Biosciences).Primary antibodies against the synthetic-KLH conjugates were made inrabbits. Secondary antibody against rabbit IgG, coupled with peroxidase,was purchased from Sigma. Both primary and secondary antibodies wereused at 1:1000 dilutions. Antibodies showed strong reactivity to asingle band on the Western Blots indicating that the antisera weremonospecific to the target peptide of interest. Antisera were alsocrossreactive with synthetic peptides conjuated to KLH.

Example 9 Nucleic Acid Identity and Structure Relatedness of IsolatedNucleic Acid Fragments

Over 100 cloned p450 fragments were sequenced in conjunction withNorthern blot analysis to determine their structural relatedness. Theapproach used utilized forward primers based either of two common p450motifs located near the carboxyl-terminus of the p450 genes. The forwardprimers corresponded to cytochrome p450 motifs FXPERF (SEQ. ID No.:372)or GRRXCP(A/G) (SEQ ID No.:373) as denoted in FIG. 4. The reverseprimers used standard primers from either the plasmid, SP6 or T7 locatedon both arms of pGEM™ plasmid, or a poly A tail. The protocol used isdescribed below.

Spectrophotometry was used to estimate the concentration of startingdouble stranded DNA following the manufacturer's protocol (BeckmanCoulter). The template was diluted with water to the appropriateconcentration, denatured by heating at 95° C. for 2 minutes, andsubsequently placed on ice. The sequencing reaction was prepared on iceusing 0.5 to 10 p.l of denatured DNAtemplate, 2 μl of 1.6 pmole of theforward primer, 8 μl of DTCS Quick Start Master Mix and the total volumebrought to 20 μl with water. The thermocycling program consisted of 30cycles of the follow cycle: 96° C. for 20 seconds, 50° C. for 20seconds, and 60° C. for 4 minutes followed by holding at 4° C.

The sequence was stopped by adding 5 μl of stop buffer (equal volume of3M NaOAc and 100 mM EDTA and 1 μl of 20 mg/ml glycogen). The sample wasprecipitated with 60 μl of cold 95% ethanol and centrifuged at 6000 gfor 6 minutes. Ethanol was discarded. The pellet was 2 washes with 200ul of cold 70% ethanol. After the pellet was dry, 40 μl of SLS solutionwas added and the pellet was resuspended. A layer of mineral oil wasover laid. The sample was then, placed on the CEQ 8000 AutomatedSequencer for further analysis.

In order to verify nucleic acid sequences, nucleic acid sequence wasre-sequenced in both directions using forward primers to the FXPERF(SEQ. ID No.:372) or GRRXCP(A/G) (SEQ. ID No.:373) region of the p450gene or reverse primers to either the plasmid or poly A tail. Allsequencing was performed at least twice in both directions.

The nucleic acid sequences of cytochrome p450 fragments were compared toeach other from the coding region corresponding to the first nucleicacid after the region encoding the GRRXCP(A/G) (SEQ. ID No.:373) motifthrough to the stop codon. This region was selected as an indicator ofgenetic diversity among p450 proteins. A large number of geneticallydistinct p450 genes, in excess of 70 genes, were observed, similar tothat of other plant species. Upon comparison of nucleic acid sequences,it was found that the genes could be placed into distinct sequencesgroups based on their sequence identity. It was found that the bestunique grouping of p450 members was determined to be those sequenceswith 75% nucleic acid identity or greater (shown in Table I). Reducingthe percentage identity resulted in significantly larger groups. Apreferred grouping was observed for those sequences with 81% nucleicacid identity or greater, a more preferred grouping 91% nucleic acididentity or greater, and a most preferred grouping for those sequences99% nucleic acid identity of greater. Most of the groups contained atleast two members and frequently three or more members. Others were notrepeatedly discovered suggesting that approach taken was able toisolated both low and high expressing mRNA in the tissue used.

Based on 75% nucleic acid identity or greater, two cytochrome p450groups were found to contain nucleic acid sequence identity topreviously tobacco cytochrome genes that genetically distinct from thatwithin the group. Group 23, showed nucleic acid identity, within theparameters used for Table I, to prior GenBank sequences of GI:1171579(CAA64635) (SEQ. ID No.:374) and GI:14423327 (or AAK62346) (SEQ. IDNo.:375) by Czernic et al and Ralston et al, respectively. GI:1171579had nucleic acid identity to Group 23 members ranging 96.9% to 99.5%identity to members of Group 23 while GI:14423327 ranged 95.4% to 96.9%identity to this group. The members of Group 31 had nucleic acididentity ranging from 76.7% to 97.8% identity to the GenBank reportedsequence of GI:14423319 (AAK62342) (SEQ. ID No.:376) by Ralston et al.None of the other p450 identity groups of Table 1 contained parameteridentity, as used in Table 1, to Nicotiana p450s genes reported byRalston et al, Czernic et al., Wang et al or LaRosa and Smigocki.

A consensus sequence with appropriate nucleic acid degenerate probescould be derived for group to preferentially identify and isolateadditional members of each group from Nicotiana plants.

TABLE I Nicotiana P450 Nucleic Acid Sequence Identity Groups GROUPFRAGMENTS 1 D58-BG7 (SEQ ID No.: 1), D58-AB1 (SEQ ID No.: 3); D58-BE4(SEQ ID No.: 7) 2 D56-AH7 (SEQ ID No.: 9); D13a-5 (SEQ ID No.: 11) 3D56-AG10 (SEQ ID No.: 13); D35-33 (SEQ ID No.: 15); D34-62 (SEQ ID No.:17) 4 D56-AA7 (SEQ ID No.: 19); D56-AE1 (SEQ ID No.: 21); 185-BD3 (SEQID No.: 143) 5 D35-BB7 (SEQ ID No.: 23); D177-BA7 (SEQ ID No.: 25);D56A-AB6 (SEQ ID No.: 27); D144-AE2 (SEQ ID No.: 29) 6 D56-AG11 (SEQ IDNo.: 31); D179-AA1 (SEQ ID No.: 33) 7 D56-AC7 (SEQ ID No.: 35); D144-AD1(SEQ ID No.: 37) 8 D144-AB5 (SEQ ID No.: 39) 9 D181-AB5 (SEQ ID No.:41); D73-Ac9 (SEQ ID No.: 43) 10 D56-AC12 (SEQ ID No.: 45) 11 D58-AB9(SEQ ID No.: 47); D56-AG9 (SEQ ID No.: 49); D56-AG6 (SEQ ID No.: 51);D35-BGll (SEQ ID No.: 53); D35-42 (SEQ ID No.: 55); D35-BA3 (SEQ ID No.:57); D34-57 (SEQ ID No.: 59); D34-52 (SEQ ID No.: 61); D34-25 (SEQ IDNo.: 63) 12 D56-AD10 (SEQ ID No.: 65) 13 56-AA11 (SEQ ID No.: 67) 14D177-BD5 (SEQ ID No.: 69); D177-BD7 (SEQ ID No.: 83) 15 D56A-AG10 (SEQID No.: 71); D58-BC5 (SEQ ID No.: 73); D58-AD12 (SEQ ID No.: 75) 16D56-AC11 (SEQ ID No.: 77); D35-39 (SEQ ID No.: 79); D58-BH4 (SEQ ID No.:81); D56-AD6 (SEQ ID No.: 87) 17 D73A-AD6 (SEQ ID No.: 89); D70A-BA11(SEQ ID No.: 91) 18 D70A-AB5 (SEQ ID No.: 95); D70A-AA8 (SEQ ID No.: 97)19 D70A-AB8 (SEQ ID No.: 99); D70A-BH2 (SEQ ID No.: 101); D70A-AA4 (SEQID No.: 103) 20 D70A-BA1 (SEQ ID No.: 105); D70A-BA9 (SEQ ID No.: 107)21 D70A-BD4 (SEQ ID No.: 109) 22 D181-AC5 (SEQ ID No.: 111); D144-AH1(SEQ ID No.: 113); D34-65 (SEQ ID No.: 115) 23 D35-BG2 (SEQ ID No.: 117)24 D73A-AH7 (SEQ ID No.: 119) 25 D58-AA1 (SEQ ID No.: 121); D185-BC1(SEQ ID No.: 133); D185-BG2 (SEQ ID No.: 135) 26 D73-AE10 (SEQ ID No.:123) 27 D56-AC12 (SEQ ID No.: 125) 28 D177-BF7 (SEQ ID No.: 127);D185-BEl (SEQ ID No.: 137); D185-BD2 (SEQ ID No.: 139) 29 D73A-AG3 (SEQID No.: 129) 30 D70A-AA12 (SEQ ID No.: 131); D176-BF2 (SEQ ID No.: 85)31 D176-BC3 (SEQ ID No.: 145) 32 D176-BB3 (SEQ ID No.: 147) 33 D186-AH4(SEQ ID No.: 5)

Example 10 Related Amino Acid Sequence Identity of Isolated Nucleic AcidFragments

The amino acid sequences of nucleic acid sequences obtained forcytochrome p450 fragments from Example 8 were deduced. The deducedregion corresponded to the amino acid immediately after the GXRXCP(A/G)(SEQ. ID No.:377) sequence motif to the end of the carboxyl-terminus, orstop codon. Upon comparison of sequence identity of the fragments, aunique grouping was observed for those sequences with 70% amino acididentity or greater. A preferred grouping was observed for thosesequences with 80% amino acid identity or greater, more preferred with90% amino acid identity or greater, and a most preferred grouping forthose sequences 99% amino acid identity of greater. The groups andcorresponding amino acid sequences of group members are shown in FIG. 1.Several of the unique nucleic acid sequences were found to have completeamino acid identity to other fragments and therefore only one memberwith the identical amino acid was reported.

The amino acid identity for Group 19 of Table II corresponded to threedistinct groups based on their nucleic acid sequences. The amino acidsequences of each group member and their identity is shown in FIG. 2.The amino acid differences are appropriated marked.

At least one member of each amino acid identity group was selected forgene cloning and functional studies using plants. In addition, groupmembers that are differentially affected by ethylene treatment or otherbiological differences as assessed by Northern and Southern analysiswere selected for gene cloning and functional studies. To assist in genecloning, expression studies and whole plant evaluations, peptidespecific antibodies will be prepared on sequence identity anddifferential sequence.

TABLE II Nicotiana p450 Amino Acid Sequence Identity Groups GROUPFRAGMENTS 1 D58-BG7 (SEQ ID No.: 2), D58-AB1 (SEQ ID No.: 4) 2 D58-BE4(SEQ ID No.: 8) 3 D56-AH7 (SEQ ID No.: 10); D13a-5 (SEQ ID No.: 12) 4D56-AG10 (SEQ ID No.: 14); D34-62 (SEQ ID No.: 18) 5 D56-AA7 (SEQ IDNo-: 20); D56-AE1 (SEQ ID No.: 22); 185 

 BD3 (SEQ ID No.: 144) 6 D35-BB7 (SEQ ID No.: 24); D177-BA7 (SEQ ID No.:26); D56A-AB6 (SEQ ID No.: 28); D144-AE2 (SEQ ID No.: 30) 7 D56-AG11(SEQ ID No.: 32); D179-AA1 (SEQ ID No.: 34) 8 D56-AC7 (SEQ ID No.: 36);D144-AD1 (SEQ ID No.: 38) 9 D144-AB5 (SEQ ID No.: 40) 10 D181-AB5 (SEQID No.: 42); D73-Ac9 (SEQ ID No.: 44) 11 D56-AC12 (SEQ ID No.: 46) 12D58-AB9 (SEQ ID No.: 48); D56-AG9 (SEQ ID No.: 50); D56 

 AG6 (SEQ ID No.: 52); D35-BG11 (SEQ ID No.: 54); D35-42 (SEQ ID No.:56); D35-BA3 (SEQ ID No.: 58); D34-57 (SEQ ID No.: 60); D34-52 (SEQ IDNo.: 62) 13 D56AD10 (SEQ ID No.: 66) 14 56-AAll (SEQ ID No.: 68) 15D177-BD5 (SEQ ID No.: 70); D177-BD7 (SEQ ID No.: 84) 16 D56A-AG10 (SEQID No.: 72); D58-BC5 (SEQ ID No.: 74); D58-AD12 (SEQ ID No.: 76) 17D56-AC11 (SEQ ID No.: 78); D56-AD6 (SEQ ID No.: 88) 18 D73A-AD6 (SEQ IDNo. 90:) 19 D70A-AB5 (SEQ ID No.: 96); D70A-AB8 (SEQ ID No.: 100);D70A-BH2 (SEQ ID No.: 102); D70A-AA4 (SEQ ID No.: 104); D70A 

 BA1 (SEQ ID No.: 106); D70A-BA9 (SEQ ID No.: 108) 20 D70A-BD4 (SEQ IDNo.: 110) 21 D181-AC5 (SEQ ID No.: 112); D144-AH1 (SEQ ID No.: 114);D34-65 (SEQ ID No.: 116) 22 D35-BG2 (SEQ ID No.: 118) 23 D73A-AH7 (SEQID No.: 120) 24 D58-AA1 (SEQ ID No.: 122); D185-BC1 (SEQ ID No.: 134);D185-BG2 (SEQ ID No.: 136) 25 D73-AE10 (SEQ ID No.: 124) 26 D56-AC12(SEQ ID No.: 126) 27 D177-BF7 (SEQ ID No.: 128); 185-BD2 (SEQ ID No.:140) 28 D73A-AG3 (SEQ ID No.: 130) 29 D70A-AA12 (SEQ ID No.: 132);D176-BF2 (SEQ ID No.: 86) 30 D176-BC3 (SEQ ID No.: 146) 31 D176-BB3 (SEQID No.: 148) 32 D186-AH4 (SEQ ID No.: 6)

Example 11 Related Amino Acid Sequence Identity of Full Length Clones

The nucleic acid sequence of full length Nicotiana genes cloned inExample 5 were deduced for their entire amino acid sequence. Cytochromep450 genes were identified by the presence of three conserved p450domain motifs, which corresponded to UXXRXXZ (SEQ. ID No.:367), PXRFXF(SEQ. ID No.:378) or GXRXC (SEQ. ID No.:379) at the carboxyl-terminuswhere U is E or K, X is any amino acid and Z is P, T, S or M. It wasalso noted that two of the clones appeared nearly complete but lackedthe appropriate stop codon, D130-AA1 and D101-BA2, but both containedall three p450 cytochrome domains. All p450 genes were characterized foramino acid identity using a BLAST program comparing their full lengthsequences to each other and to known tobacco genes. The program used theNCBI special BLAST tool (Align two sequences (bl2seq), available atncbi.nlm.nih.gov/blast/bl2seq/b12.html on the World Wide Web). Twosequences were aligned under BLASTN without filter for nucleic acidsequences and BLASTP for amino acid sequences. Based on their percentageamino acid identity, each sequence was grouped into identity groupswhere the grouping contained members that shared at least 85% identitywith another member. A preferred grouping was observed for thosesequences with 90% amino acid identity or greater, a more preferredgrouping had 95% amino acid identity or greater, and a most preferredgrouping had those sequences 99% amino acid identity or greater. Usingthese criteria, 25 unique groups were identified and are depicted inTable III.

Within the parameters used for Table III for amino acid identity, threegroups were found to contain greater than 85% or greater identity toknown tobacco genes. Members of Group 5 had up to 96% amino acididentity for full length sequences to prior GenBank sequences ofGI:14423327 (or AAK62346) (SEQ. ID No.:375) by Ralston et al. Group 23had up to 93% amino acid identity to GI:14423328 (or AAK62347) (SEQ. IDNo.:380) by Ralston et al. and Group 24 had 92% identity to GI:14423318(SEQ. ID No.:381) (or AAK62343) (SEQ. ID No.:382) by Ralston et al.

TABLE III Amino Acid Sequence Identity Groups of Full Length NicotianaD450 Genes 1 D208-AD9 (SEQ. ID. No. 224); D120-AH4 (SEQ. ID. No. 180);D121-AA8 (SEQ. ID. No. 182), D122-AF10 (SEQ. ID. No. 184); D103-AH3(SEQ. ID. No. 222); D208-AC8 (SEQ. ID. No. 218); D-235-ABI (SEQ. ID. No.246) 2 D244-AD4 (SEQ. ID. No. 250); D244-AB6 (SEQ. ID. No. 274);D285-AA8; D285-AB9; D268-AE2 (SEQ. ID. No. 270) 3 D100A-AC3 (SEQ. ID.No. 168); D100A-BE2 4 D205-BE9 (SEQ. ID. No. 276); D205-BG9 (SEQ. ID.No. 202); D205-AH4 (SEQ. ID. No. 294) 5 D259-AB9 (SEQ. ID. No. 260);D257-AE4 (SEQ. ID. No. 268); D147-AD3 (SEQ. ID. No. 194) 6 D249-AE8(SEQ. ID. No. 256); D-248-AA6 (SEQ. ID. No. 254) 7 D233-AG7 (SEQ. ID.No. 266; D224-BD11 (SEQ. ID. No. 240); DAF10 8 D105-AD6 (SEQ. ID. No.172); D215-AB5 (SEQ. ID. No. 220); D135-AE1 (SEQ. ID. No. 190) 9D87A-AF3 (SEQ. ID. No. 216), D210-BD4 (SEQ. ID. No. 262) 10 D89-AB1(SEQ. ID. No. 150); D89-AD2 (SEQ. ID. No. 152); 163-AG11 (SEQ. ID. No.198); 163-AF12 (SEQ. ID. No. 196) 11 D267-AF10 (SEQ. ID. No. 296);D96-AC2 (SEQ. ID. No. 204); D207-AB4 (SEQ. ID. No. 206); D207-AC4 (SEQ.ID. No. 208) 12 D98-AG1 (SEQ. ID. No. 164); D98-AA1 (SEQ. ID. No. 162)13 D209-AA12 (SEQ. ID. No. 212); D209-AA11; D209-AH10 (SEQ. ID. No.214); D209-AH12 (SEQ. ID. No. 232); D90a-BB3 (SEQ. ID. No. 154) 14D129-AD10 (SEQ. ID. No. 188); D104A-AE8 (SEQ. ID. No. 170) 15 D228-AH8(SEQ. ID. No. 244); D228-AD7 (SEQ. ID. No. 241), D250-AC11 (SEQ. ID. No.258); D247-AH1 (SEQ. ID. No. 252) 16 D128-AB7 (SEQ. ID. No. 186);D243-AA2 (SEQ. ID. No. 248); D125-AF11 (SEQ. ID. No. 228) 17 D284-AH5(SEQ. ID. No. 298); D110-AF12 (SEQ. ID. No. 176) 18 D221-BB8 (SEQ. ID.No. 234) 19 D222-BH4 (SEQ. ID. No. 236) 20 D134-AE11 (SEQ. ID. No. 230)21 D109-AH8 (SEQ. ID. No. 174) 22 D136-AF4 (SEQ. ID. No. 278) 23D237-AD1 (SEQ. ID. No. 226) 24 D112-AA5 (SEQ. ID. No. 178) 25 D283-AC1(SEQ. ID. No. 272)

The full length genes were further grouped based on the highly conversedamino acid homology between UXXRXXZ (SEQ. ID No.:367) p450 domain andGXRXC (SEQ. ID No.:379) p450 domain near the end the carboxyl-terminus.As shown in FIG. 3, individual clones were aligned for their sequencehomology between the conserved domains relative to each other and placedin distinct identity groups. In several cases, although the nucleic acidsequence of the clone was unique, the amino acid sequence for the regionwas identical. The preferred grouping was observed for those sequenceswith 90% amino acid identity or greater, a more preferred group had 95%amino acid identity or greater, and a most preferred grouping had thosesequences 99% amino acid identity of greater. The final grouping wassimilar to that based on the percent identity for the entire amino acidsequence of the clones except for Group 17 (of Table III) which wasdivided into two distinct groups.

Within the parameters used for amino acid identity in Table IV, threegroups were found to contain 90% or greater identity to known tobaccogenes. Members of Group 5 had up to 93.4% amino acid identity for fulllength sequences to prior GenBank sequences of GI:14423326 (AAK62346)(SEQ. ID No.:383) by Ralston et al. Group 23 had up to 91.8% amino acididentity to GI:14423328 (or AAK62347) (SEQ. ID No.:380) by Ralston etal. and Group 24 had 98.8% identity to GI:14423318 (or AAK62342) (SEQ.ID No.:381) by Ralston et al.

TABLE IV Amino Acid Sequence Identity Groups of Regions betweenConserved Domains of Nicotiana p450 Genes 1 1 D208-AD9 (SEQ. ID. No.224); D120-AH4 (SEQ. ID. No. 180); D121-AA8 (SEQ. ID. No. 182),D122-AF10 (SEQ. ID. No. 184); D103-AH3 (SEQ. ID. No. 222); D208-ACS(SEQ. ID. No. 218); D-235-ABI (SEQ. ID. No. 246) 2 D244-AD4 (SEQ. ID.No. 250); D244-AB6 (SEQ. ID. No. 274); D285-AA8; D285-AB9; D268-AE2(SEQ. ID. No. 270) 3 D100A-AC3 (SEQ. ID. No. 168); D100A-BE2 4 D205-BE9(SEQ. ID. No. 276); D205-BG9 (SEQ. ID. No. 202); D205-AH4 (SEQ. ID. No.294) 5 D259-AB9 (SEQ. ID. No. 260); D257-AE4 (SEQ. ID. No. 268);D147-AD3 (SEQ. ID. No. 194) 6 D249-AE8 (SEQ. ID. No. 256); D-248-AA6(SEQ. ID. No. 254) 7 D233-AG7 (SEQ. ID. No. 266; D224-BD11 (SEQ. ID. No.240); DAF10 8 D105-AD6 (SEQ. ID. No. 172); D215-AB5 (SEQ. ID. No. 220);D135-AE1 (SEQ. ID. No. 190) 9 D87A-AF3 (SEQ. ID. No. 216), D210-BD4(SEQ. ID. No. 262) 10 D89-AB1 (SEQ. ID. No. 150); D89-AD2 (SEQ. ID. No.152); 163-AG11 (SEQ. ID. No. 198); 163-AF12 (SEQ. ID. No. 196) 11D267-AF10 (SEQ. ID. No. 296); D96-AC2 (SEQ. ID. No. 160); D96-A136 (SEQ.ID. No. 158); D207-AA5 (SEQ. ID. No. 204); D207-AB4 (SEQ. ID. No. 206);D207-AC4 (SEQ. ID. No. 208) 12 D98-AG1 (SEQ. ID. No. 164); D98-AA1 (SEQ.ID. No. 162) 13 D209-AA12 (SEQ. ID. No. 212); D209-AA11; D209-AH10 (SEQ.ID. No. 214); D209-AH12 (SEQ. ID. No. 232); D90a-BB3 (SEQ. ID. No. 154)14 D129-AD10 (SEQ. ID. No. 188); D104A-AE8 (SEQ. ID. No. 170) 15D228-AH8 (SEQ. ID. No. 244); D228-AD7 (SEQ. ID. No. 241), D250-AC11(SEQ. ID. No. 258); D247-AH1 (SEQ. ID. No. 252) 16 D128-AB7 (SEQ. ID.No. 186); D243-AA2 (SEQ. ID. No. 248); D125-AF11 (SEQ. ID. No. 228) 17D284-AH5 (SEQ. ID. No. 298); D110-AF12 (SEQ. ID. No. 176) 18 D221-BB8(SEQ. ID. No. 234) 19 D222-BH4 (SEQ. ID. No. 236) 20 D134-AE11 (SEQ. ID.No. 230) 21 D109-AH8 (SEQ. ID. No. 174) 22 D136-AF4 (SEQ. ID. No. 278)23 D237-AD1 (SEQ. ID. No. 226) 24 D112-AA5 (SEQ. ID. No. 178) 25D283-AC1 (SEQ. ID. No. 272) 26 D110-AF12 (SEQ. ID. No. 176)

Example 12 Nicotiana Cytochrome P450 Clones Lacking One or More of theTobacco Cytochrome P450 Specific Domains

Four clones had high nucleic acid homology, ranging 90% to 99% nucleicacid homology, to other tobacco cytochrome genes reported in Table III.The four clones included D136

AD5, D138-AD12, D243-AB3 and D250-AC11. However, due to a nucleotideframeshift these genes did not contain one or more of three C-terminuscytochrome p450 domains and were excluded from identity groups presentedin Table III or Table IV.

The amino acid identity of one clone, D95-AG1, did not contain the thirddomain, GXRXC, used to group p450 tobacco genes in Table III or TableIV. The nucleic acid homology of this clone had low homology to othertobacco cytochrome genes. This clone represents a novel and differentgroup of cytochrome p450 genes in Nicotiana.

Example 13 Use of Nicotiana Cytochrome P450 Fragments and Clones inAltered Regulation of Tobacco Properties

The use of tobacco p450 nucleic acid fragments or whole genes are usefulin identifying and selecting those plants that have altered tobaccophenotypes or tobacco constituents and, more importantly, alteredmetabolites. Transgenic tobacco plants are generated by a variety oftransformation systems that incorporate nucleic acid fragments or fulllength genes, selected from those reported herein, in orientations foreither down-regulation, for example anti-sense orientation, orover-expression for example, sense orientation. For over-expression tofull length genes, any nucleic acid sequence that encodes the entire ora functional part or amino acid sequence of the full-length genesdescribed in this invention are desired that are effective forincreasing the expression of a certain enzyme and thus resulting inphenotypic effect within Nicotiana. Nicotiana lines that are homozygouslines are obtained through a series of backcrossing and assessed forphenotypic changes including, but not limited to, analysis of endogenousp450 RNA, transcripts, p450 expressed peptides and concentrations ofplant metabolites using techniques commonly available to one havingordinary skill in the art. The changes exhibited in the tobacco plansprovide information on the functional role of the selected gene ofinterest or are of a utility as a preferred Nicotiana plant species.

Example 14 Identification of Genes Induced in Ethylene Treated ConverterLines

High density oligonucleotide array technology, Affymetrix GENECHIP® genearray (Affymetrix Inc., Santa Clara, Calif.), was used for quantitativeand highly parallel measurements of gene expression. In using thistechnology, nucleic acid arrays were fabricated by direct synthesis ofoligonucleotides on a solid surface. This solid-phase chemistry is ableto produce arrays containing hundreds of thousands of oligonucleotideprobes packed at extremely high densities on a chip referred to asGENECHIP® gene array. Thousands of genes can be simultaneously screenedfrom a single hybridization. Each gene is typically represented by a setof 11-25 pairs of probes depending upon size. The probes are designed tomaximize sensitivity, specificity, and reproducibility, allowingconsistent discrimination between specific and background signals, andbetween closely related target sequences.

Affymetrix GENECHIP® gene array hybridization experiments involve thefollowing steps: design and production of arrays, preparation offluorescently labeled target from RNA isolated from the biologicalspecimens, hybridization of the labeled target to the GENECHIP® genearray, screening the array, and analysis of the scanned image andgeneration of gene expression profiles.

A. Designing and Custom Making Affymetrix GENECHIP® Gene Array

A GENECHIP® CustomExpress Advantage Array gene array was custom made byAffymetrix Inc. (Santa Clara, Calif.). Chip size was 18 micron and arrayformat was 100-2187 that can accommodate 528 probe sets (11,628 probes).Except for GenBank derived nucleic acid sequences, all sequences wereselected from our previously identified tobacco clones and all probeswere custom designed. A total of 400 tobacco genes or fragments wereselected to be included on the GENECHIP® gene array. The sequences ofoligonucleotides selected were based on unique regions of the 3′ end ofthe gene. The selected nucleic acid sequences consisted of 56 fulllength p450 genes and 71 p450 fragments that were cloned from tobacco,described in (patent applications). Other tobacco sequences included 270tobacco ESTs which were generated from suppression subtraction libraryusing Clontech SSH kit(BD Biosciences, Palo Alto, Calif.). Among thesegenes, some oligonucleotide sequences were selected from cytochrome P450genes listed in GenBank. Up to 25 probes were used for each full lengthgene and 11 probes for each fragment. A reduced number of probes wereused for some clones due to the lack of unique, high quality probes.Appropriate control sequences were also included on the GENECHIP® genearray.

The probe Arrays were 25-mer oligonucleotides that were directlysynthesized onto a glass wafer by a combination of semiconductor-basedphotolithography and solid phasechemical synthesis technologies. Eacharray contained up to 100,000 different oligonucleotide probes. Sinceoligonucleotide probes are synthesized in known locations on the array,the hybridization patterns and signal intensities can be interpreted interms of gene identity and relative expression levels by the AffymetrixMICROARRAY SUITE® software. Each probe pair consists of a perfect matcholigonucleotide and a mismatch oligonucleotide. The perfect match probehas a sequence exactly complimentary to the particular gene and thusmeasures the expression of the gene. The mismatch probe differs from theperfect match probe by a single base substitution at the center baseposition, which disturbs the binding of the target gene transcript. Themismatch produces a nonspecific hybridization signal or backgroundsignal that was compared to the signal measured for the perfect matcholigonucleotide.

B. Sample Preparation

Hybridization experiments were conducted by Genome Explorations, Inc.(Memphis, Tenn.). The RNA samples used in hybridization consisted of sixpairs of nonconverter/converter isogenic lines that were induced byethylene treatments. Samples included one pair of 4407-25/4407-33non-treated burly tobacco samples, three pairs of ethylene treated4407-25/4407

33 samples, one pair of ethylene treated dark tobacco NL Madole/181 andone pair of ethylene treated burly variety PBLB01/178. Ethylenetreatment was as described in Example 1.

Total RNA was extracted from above mentioned ethylene treated andnon-treated leaves using a modified acid phenol and chloroformextraction protocol. Protocol was modified to use one gram of tissuethat was ground and subsequently vortexed in 5 ml of extraction buffer(100 mM Tris-HCl, pH 8.5; 200 mM NaCl; 10 mM EDTA; 0.5% SDS) to which 5ml phenol (pH5.5) and 5 ml chloroform was added. The extracted samplewas centrifuged and the supernatant was saved. This extraction step wasrepeated 2-3 more times until the supernatant appeared clear.Approximately 5 ml of chloroform was added to remove trace amounts ofphenol. RNA was precipitated from the combined supernatant fractions byadding a 3-fold volume of ETOH and 1/10 volume of 3M NaOAc (pH5.2) andstoring at −20° C. for 1 hour. After transferring to a Corex glasscontainer the RNA fraction was centrifuged at 9,000 RPM for 45 minutesat 4° C. The pellet was washed with 70% ethanol and spun for 5 minutesat 9,000 RPM at 4° C. After drying the pellet, the pelleted RNA wasdissolved in 0.5 ml RNase free water. The pelleted RNA was dissolved in0.5 ml RNase free water. The quality and quantity of total RNA wasanalyzed by denatured formaldehyde gel and spectrophotometer,respectively. The total RNA samples with 3-5 μg/ul were sent to Genomeexplorations, inc. to do the hybridization.

C. Hybridization, Detection and Data Output

The preparation of labeled cRNA material was performed as follows. Firstand second strand cDNA were synthesized from 5-15 jag of total RNA usingthe Superscript Double-Stranded cDNA Synthesis Kit (Gibco LifeTechnologies) and oligo-dT24-T7 (5′

GGC CAG TGA ATT GTA ATA CGA CTC ACT ATA GGG AGG CGG-3′) primer (SEQ. IDNo.:384) according to the manufacturer's instructions.

The cRNA was concurrently synthesized and labeled with biotinylated UTPand CTP by in vitro transcription using the T7 promoter coupled doublestranded cDNA as template and the T7 RNA Transcript Labeling Kit (ENZODiagnostics Inc.). Briefly, double stranded cDNA synthesized from theprevious steps were washed twice with 70% ethanol and resuspended in 22pl Rnase-free H2O. The cDNA was incubated with 4 dal of 10× eachReaction Buffer, Biotin Labeled Ribonucleotides, DTT, Rnase InhibitorMix and 2 pl 20X T7 RNA Polymerase for 5 hr at 37° C. The labeled cRNAwas separated from unincorporated ribonucleotides by passing through aCHROMA SPIN-100 column (Clontech) and precipitated at −20° C. for 1 hrto overnight.

Oligonucleotide array hybridization and analysis were performed asfollows. The cRNA pellet was resuspended in 10 μl Rnase-free H2O and 10μg was fragmented by heat and ion-mediated hydrolysis at 95° C. for 35rains in 200 mM Tris-

acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc. The fragmented cRNA washybridized for 16 hr at 45° C. to HG U95Av2 oligonucleotide arrays(Affymetrix) containing −12,500 full length annotated genes togetherwith additional probe sets designed to represent EST sequences. Arrayswere washed at 25° C. with 6×SSPE (0.9M NaCl, 60 mMNaH2PO4, 6 mMEDTA+0.01% Tween 20) followed by a stringent wash at 50° C. with 100 mMMES, 0.1M [Na+], 0.01% Tween 20. The arrays were stained withphycoerythrein conjugated streptavidin (Molecular Probes) and thefluorescence intensities were determined using a laser confocal scanner(Hewlett-Packard). The scanned images were analyzed using Microarraysoftware (Affymetrix). Sample loading and variations in staining werestandardized by scaling the average of the fluorescent intensities ofall genes on an array to constant target intensity (250) for all arraysused. Data Analysis was conducted using Microarray Suite 5.0(Affymetrix) following user guidelines. The signal intensity for eachgene was calculated as the average intensity difference, represented by[E(PM−MM)/(number of probe pairs)], where PM and MM denote perfect-matchand mismatch probes.

D. Data Analysis and Results

Twelve sets of hybridizations were successful as evidenced by theExpression Report generated using detection instruments from GenomeExplorations. The main parameters on the report included Noise, Scalefactor, background, total probe sets, number and percentage of presentand absent probe sets, signal intensity of housekeeping controls. Thedata was subsequently analyzed and presented using software GCOS incombination of other Microsoft software. Signal comparison betweentreatment pairs was analyzed. Overall data for all respective probescorresponding to genes and fragments of each different treatmentincluding replications were compiled and compiled expression data suchas call of the changes and signal log 2 ratio changes were analyzed.

A typical application of GENECHIP® gene array technology is findinggenes that are differentially expressed in different tissues. In thepresent application, genetic expression variations caused by ethylenetreatment were determined for pairs of converter and nonconvertertobacco lines that included a 4407-25/4407-33 burley variety, PBLBO1/178burley variety, and a NL Madole/181 dark variety. These analysesdetected only those genes whose expression is significantly altered dueto biological variation. These analyses employed the Fold change (signalratio) as a major criterion to identify induced genes. Other parameters,such as signal intensity, present/absent call, were also taken intoconsideration.

After analyzing the data for expression differences in converter andnonconverter pairs of samples for approximately 400 genes, the resultsbased on the signal intensities showed that only two genes, D121-AA8,and D120-AH4 and one fragment, D35-BG11, that is partial fragment ofD121-AA8, had reproducible induction in ethylene treated converter linesversus non-converter lines. To illustrate the differential expression ofthese genes, the data was represented as follows. As shown in Table V,the signal of a gene in a converter line, for example, burley tobaccovariety, 4407-33, was determined as ratio to the signal of a relatednonconverter isogenic line, 4407-25. Without ethylene treatment, theratio of converter to nonconverter signals for all genes approached1.00. Upon ethylene treatment, two genes, D121-AA8 and D120-AH4, wereinduced in converter lines relative to non-converter line as determinedby three independent analyses using isogenic burley lines. These geneshave very high homology to each other, approximately 99.8% or greaternucleic acid sequence homology. As depicted in Table V, their relativehybridization signals in converter varieties ranged from approximately 2to 12 fold higher in converter lines than the signals in theirnon-converter counterparts. In comparison, two actin-like controlclones, internal controls, were found not to be induced in converterlines based on their normalized ratios. In addition, a fragment(D35-BG11), whose sequence in coding region is entirely contained inboth D121-AA8 and D120-AH4 genes, was highly induced in the same samplesof paired isogenic converter and nonconverter lines. Another isogenicpair of burley tobacco varieties, PBLB01 and 178, was shown to have thesame genes, D121-AA8 and D120-AH4, induced in converter samples underethylene induction. Furthermore, D121-AA8 and D120-AH4 genes werepreferentially induced in converter lines of isogenic dark tobaccopairs, NL Madole and 181, demonstrating that ethylene induction of thesegenes in converter lines was not limited to burley tobacco varieties. Inall cases, the D35-BG11 fragment was the most highly induced inconverter relative to nonconverter paired lines.

TABLE V A Comparison of Clone Induction in Ethylene Treated Converterand Non- Converter Lines Ethylene Ethylene Ethylene No Treated BurleyTreated Treated Ethylene Treated Ethylene Treatment Exp 1 Burley Exp 2Burley Exp 3 Burley Exp 4 Treated Dark Clones 33:25 33:25 Et:No* 33.25Et:No 33.25 Et:No 33:25 Et:No 181:NL Et:No Induced Ratio Ratio RatioRatio Ratio Ratio Ratio Ratio Ratio Ratio Ratio D121-AA8 1.03 2.20 2.1413.25 12.90 5.31 5.15 12.56 12.19 17.06 16.60 D120-AH4 1.44 2.74 1.9018.33 12.74 4.13 2.87 10.87 7.55 11.76 8.17 Control Actin-Like I 1.181.17 0.99 0.88 0.74 0.86 0.73 0.67 0.57 1.20 1.02 (5′) Actin-Like I 1.091.23 1.12 0.89 0.81 1.18 0.11 0.86 0.79 1.02 0.93 (3′) *normalized Ratio

Example 15 Ethylene Induction of Microsomal Nicotine Demethylase inTobacco Converter Lines

Biochemical analyses of demethylase enzymatic activity in microsomalenriched fractions of ethylene treated and non-treated pairs ofconverter and non-converter tobacco lines were performed as follows.

A. Preparation of Microsomes

Microsomes were isolated at 4° C. Tobacco leaves were extracted in abuffer consisting of 50 mMN-(2-hydrooxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), pH7.5, 3 mM DL-Dithiothreitol (DTT) and Protease Inhibitor Cocktail(Roche) at 1 tablet/50 ml. The crude extract was filtered through fourlayers of cheesecloth to remove undisrupted tissue, and the filtrate wascentrifuged for 20 min at 20,000×g to remove cellular debris. Thesupernatant was subjected to ultracentrifugation at 100,000×g for 60 minand the resultant pellet contained the microsomal fraction. Themicrosomal fraction was suspended in the extraction buffer and appliedto an ultracentrifugation step where a discontinuous sucrose gradient of0.5 M sucrose in the extraction buffer was used. The purified microsomeswere resuspended in the extraction buffer supplemented with 10% (w/v)glycerol as cryoprotectant. Microsomal preparations were stored in aliquid nitrogen freezer until use.

B. Protein Concentration Determination

Microsomal proteins were precipitated with 10% Trichloroacetic Acid(TCA) (w/v) in acetone, and the protein concentrations of microsomeswere determined using RC DC Protein Assay Kit (BIO-RAD) following themanufacturer's protocol.

3) Nicotine Demethylase Activity Assay DL-Nicotine (Pyrrolidine-2-¹⁴C)was obtained from Moravek Biochemicals and had a specific activity of 54mCi/mmol. Chlorpromazine (CPZ) and oxidized cytochrome c (cyt. C), bothP450 inhibitors, were purchased from Sigma. Reduced form of nicotinamideadenine dinucleotide phosphate (NADPH) is the typical electron donor forcytochrome P450 via the NADPH:cytochrome P450 reductase. NADPH wasomitted for control incubation. Routine enzyme assay consisted ofmicrosomal proteins (around 2 mg/ml), 6 mM NADPH, 55 μM 14C labelednicotine. The concentration of CPZ and Cyt. C, when used, was 1 mM and100 μM, respectively. The reaction was carried at 25° C. for 1 hour andwas stopped with addition of 300 μl methanol to each 25 μl reactionmixture. After spinning, 20 μl of the methanol extract was separatedwith a reverse-phase High Performance Liquid Chromatography (HPLC)system (Agilent) using an Inertsil ODS-3 3μ (150×4.6 mm) column fromVarian. The isocratic mobile phase was the mixture of methanol and 50 mMpotassium phosphate buffer, pH 6.25, with ratio of 60:40 (v/v) and theflow rate was 1 ml/min. The nornicotine peak, as determined bycomparison with authentic non-labeled nornicotine, was collected andsubjected to 2900 tri-carb Liquid Scintillation Counter (LSC) (PerkinElmer) for quantification. The activity of nicotine demethylase iscalculated based on the production of 14C labeled nornicotine over 1hour incubation.

Samples were obtained from pairs of Burley converter (line 4407-33) andnon-converter (line 4407-25) tobacco lines that were ethylene treated ornot. All untreated samples did not have any detectable microsomalnicotine demethylase activity. In contrast, microsomal samples obtainedfrom ethylene treated converter lines were found to contain significantlevels of nicotine demethylase activity. The nicotine demethylaseactivity was shown to be inhibited by P450 specific inhibitorsdemonstrating the demethylase activity was consistent to a P450microsomal derived enzyme.

A typical set of enzyme assay results obtained for the burley convertertobacco line is shown in the Table VI. In contrast, sample derived fromethylene treated nonconverter tobacco did not contain any nicotinedemethylase activity. These results demonstrated that nicotinedemethylase activity was induced upon treatment with ethylene inconverter lines but not in the corresponding isogenic nonconverter line.Similar results were obtained for an isogenic dark tobacco variety pair,where microsomal nicotine demethylase activity was induced in converterlines and not detectable in nonconverter paired lines. Together theseexperiments demonstrated that microsomal nicotine demethylase activityis induced upon ethylene treatment in converter lines while not inpaired isogenic nonconverter lines. Those genes that are P450 derivedgenes and are preferentially induced in converter lines relative topaired non-converter lines are candidate genes to encode the nicotinedemethylase enzyme.

TABLE VI DEMETHYLASE ACTIVITY IN MICROSOMES OF ETHYLENE 20INDUCED BURLEYCONVERTER AND NON CONVERTER LINES Microsomes + Microsomes + with 100 μMMicrosomes − Sample Microsomes 1 mM chlorpromazine cytochrome C NADPHConverter 8.3 ± 0.4 pkat/mg 0.01 ± 0.01 pkat/mg 0.2 ± 0.2 pkat/mg 0.4 ±0.4 pkat/mg protein protein protein protein Non-Converter Not DetectedNot Detected Not Detected Not Detected

Example 16 Functional Identification of D121-AA8 as Nicotine Demethylase

The function of the candidate clone (D121-AA8), was confirmed as thecoding gene for nicotine demethylase, by assaying enzyme activity ofheterologously expressed P450 in yeast cells.

1. Construction of Yeast Expression Vector

The putative protein-coding sequence of the P450-encoding cDNA (121AA8),was cloned into the yeast expression vector pYeDP60. Appropriate BamHIand MfeI sites (underlined) were introduced via PCR primers containingthese sequences either upstream of the translation start coden (ATG) ordownstream of the stop coden (TAA). The MfeI on the amplified PCRproduct is compatible with the EcoRI site on the vector. The primersused to amplify the 121AA8 cDNA were5′-TAGCTACGCGGATCCATGCTTTCTCCCATAGAAGCC-3′ (SEQ. ID No.:385) and5′-CTGGATCACAATTGTTAGTGATGGTGATGGTGATGCGATCCTCTATAAAGCTCAG GTGCCAGGC-3′(SEQ. ID No.:386). A segment of sequence coding nine extra amino acidsat the C-terminus of the protein, including six histidines, wasincorporated into the reverse primer. This facilitates the expression of6×His tagged P450 upon induction. PCR products were ligated into pYeDP60vector after enzyme digestions in the sense orientation with referenceto the GAL10-CYC1 promoter.

Constructs were verified by enzyme restrictions and DNAsequencing.

2. Yeast Transformation

The WAT11 yeast line, modified to express Arabidopsis NADPH-cytochromeP450 reductase ATR1, was transformed with the construct pYeDP60-P450cDNA plasmids. Fifty micro-liter of WAT11 yeast cell suspension wasmixed with ˜1 μg plasmid DNA in a cuvette with 0.2-cm electrode gap. Onepulse at 2.0 kV was applied by an Eppendorf electroporator (Model 2510).Cells were spread onto SGI plates (5 g/L bactocasamino acids, 6.7 g/Lyeast nitrogen base without amino acids, 20 g/L glucose, 40 mg/LDL-tryptophan, 20 g/L agar). Transformants were confirmed by PCRanalysis performed directly on randomly selected colonies.

3. P450 Expression in Transformed Yeast Cells

Single yeast colonies were used to inoculate 30 mL SGI media (5 g/Lbactocasamino acids, 6.7 g/L yeast nitrogen base without amino acids, 20g/L glucose, 40 mg/L DL-tryptophan) and grown at 30° C. for about 24hours. An aliquot of this culture was diluted 1:50 into 1000 mL of YPGEmedia (10 g/L yeast extract, 20 g/L bacto peptone, 5 g/L glucose, 30ml/L ethanol) and grown until glucose was completely consumed asindicated by the colorimetric change of a Diastix urinalysis reagentstrip (Bayer, Elkhart, Ind.). Induction of cloned P450 was initiated byadding DL-galactose to a final concentration of 2%. The cultures weregrown for an additional 20 hours before used for in vivo activity assayor for microsome preparation.

WAT11 yeast cells expressing pYeDP60-CYP71D20 (a P450 catalyzing thehydroxylation of 5-epi-aristolochene and 1

deoxycapsidiol in Nicotiana tabacum) were used as control for the P450expression and enzyme activity assays.

4. In Vivo Enzyme Assay

The nicotine demethylase activity in the transformed yeast cells wereassayed by feeding of yeast culture with DL Nicotine(Pyrrolidine-2-¹⁴C). To 75 μl of the galactose induced culture ¹⁴Clabeled nicotine (54 mCi/mmol) was added to a final concentration of 55μM. The assay culture was incubated with shaking in 14 ml polypropylenetubes for 6 hours and was extracted with 900 μl methanol. Afterspinning, 20 μA of the methanol extract was separated with an rp-HPLCand the nornicotine fraction was quantitated by LSC.

The control culture of WAT11 (pYeDP60-CYP71D20) did not convert nicotineto nornicotine, showing that the WAT11 yeast strain does not containendogenous enzyme activities that can catalyze the step of nicotinebioconversion to nornicotine. In contrast, yeast expressing 121AA8 geneproduced detectable amount of nornicotine, indicating the nicotinedemethylase activity of this P450 enzyme.

5. Yeast Microsome Preparation

After induction by galactose for 20 hours, yeast cells were collected bycentrifugation and washed twice with TES-M buffer (50 mM Tris-HCl, pH7.5, 1 mM EDTA, 0.6 M sorbitol, 10 mM 2-mercaptoethanol). The pellet wasresuspended in extraction buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.6M sorbitol, 2 mM 2-mercaptoethanol, 1% bovine serum album, ProteaseInhibitor Cocktail (Roche) at 1 tablet/50 ml). Cells were then brokenwith glass beads (0.5 mm in diameter, Sigma). Cell extract wascentrifuged for 20 min at 20,000×g to remove cellular debris. Thesupernatant was subjected to ultracentrifugation at 100,000×g for 60 minand the resultant pellet contained the microsomal fraction. Themicrosomal fraction was suspended in TEG-M buffer (50 mM Tris

HCl, pH 7.5, 1 mM EDTA, 20% glycerol and 1.5 mM 2

mercaptoethanol) at protein concentration of 1 mg/mL. Microsomalpreparations were stored in a liquid nitrogen freezer until use.

6. Enzyme Activity Assay in Yeast Microsomal Preparations

Nicotine demethylase activity assays with yeast microsomal preparationswere performed in the same way as with microsomal preparations fromtobacco leaves (EXAMPLE 15) except that the protein concentrations wereconstant at 1 mg/mL.

Microsomal preparations from control yeast cells expressing CYP71D20 didnot have any detectable microsomal nicotine demethylase activity. Incontrast, microsomal samples obtained from yeast cells expressing 121AA8gene showed significant levels of nicotine demethylase activity. Thenicotine demethylase activity had requirement for NADPH and was shown tobe inhibited by P450 specific inhibitors, consistent to the P450 beinginvestigated. A typical set of enzyme assay results obtained for theyeast cells is shown in the Table VII.

TABLE VII DEMETHYLASE ACTIVITY IN MICROSOMES OF YEAST CELLS EXPRESSING121AA8 AND CONTROL P450 Microsomes + Microsomes + with 100 μM Microsomes− Sample Microsomes 1 mM chlorpromazine cytochrome C NADPH D121-AA8 10.8± 1.2* pkat/mg 1.4 ± 1.3 pkat/mg 2.4 ± 0.7 pkat/mg 0.4 ± 0.1 pkat/mgprotein protein protein protein Control Not Detected Not Detected NotDetected Not Detected (CYP71D20) *Average results of 3 replicates.Together these experiments demonstrated that the cloned full length geneD121-AA8 encodes cytochrome P450 protein that 20 catalyzes theconversion of nicotine to nornicotine when expressed in yeast.

Numerous modifications and variations in practice of the invention areexpected to occur to those skilled in the art upon consideration of theforegoing detailed description of the invention. Consequently, suchmodifications and variations are intended to be included within thescope of the following claims.

1. An isolated nucleic acid molecule, wherein said nucleic acid moleculehas at least 91% sequence identity to SEQ. ID. No.:
 179. 2. The isolatednucleic acid molecule of claim 1, wherein said nucleic acid molecule isSEQ. ID. No.:
 179. 3. A transgenic tobacco plant transformed with thenucleic acid molecule of claim
 1. 4. The transgenic tobacco plant ofclaim 3, wherein said nucleic acid molecule is SEQ ID NO:
 179. 5. Amethod of producing a transgenic tobacco plant, said method comprising(i) operably linking the nucleic acid molecule of claim 1 with apromoter functional in said plant to create a plant transformationalvector; (ii) transforming a plurality of tobacco plant cells with saidplant transformational vector of step (i); (iii) regenerating aplurality of transformed tobacco plants from said transformed plantcells, and (iv) identifying at least one of said transformed tobaccoplants expressing said nucleic acid.
 6. The method of claim 5, whereinsaid nucleic acid molecule is in an antisense orientation.
 7. The methodof claim 5, wherein said nucleic acid molecule is in a senseorientation.
 8. The method of claim 5, wherein said nucleic acidmolecule is expressed as a double stranded RNA molecule.
 9. The methodof claim 5, wherein said nucleic acid molecule is SEQ ID NO:
 179. 10. Amethod of decreasing nornicotine levels in a tobacco plant, said methodcomprising the steps of: (i) operably linking the nucleic acid moleculeof claim 1 with a promoter functional in said plant to create a planttransformational vector; (ii) transforming a plurality of tobacco plantcells with said plant transformational vector of step (i); (iii)regenerating a plurality of transformed tobacco plants from saidtransformed plant cells; and (iv) selecting at least one of saidtransformed tobacco plants in which nornicotine levels are decreased.11. The method of claim 10, wherein said nucleic acid molecule is in anantisense orientation.
 12. The method of claim 10, wherein said nucleicacid molecule is in a sense orientation.
 13. The method of claim 10,wherein said nucleic acid molecule is expressed as a double stranded RNAmolecule.
 14. The method of claim 10, wherein said nucleic acid moleculeis SEQ ID NO: 179.